The present invention relates to obtaining a natural mixture of conjugated equine estrogens which is depleted in non-conjugated lipophilic compounds selected from the group consisting of non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, in particular androstane and pregnane steroids, and comparable non-conjugated compounds.
Estrogens are used in medicine for hormone replacement therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of disorders of the climacteric period which occur in women after natural or artificial menopause. In this case, natural mixtures of conjugated estrogens such as are found in the urine of pregnant mares, hereafter referred to as natural mixtures of conjugated equine estrogens, have proved particularly effective and readily compatible.
The dissolved solids content in the urine of pregnant mares (=pregnant mares' urine, abbreviated hereafter as “PMU”) can naturally fluctuate within wide ranges, and may generally lie in a range of 40 to 90 g dry matter per liter. In addition to urea and other usual urine contents, the solids content of the PMU contains phenolic constituents in quantities of about 2 to 5% by weight relative to the dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[(3-hydroxyphenyl)methyl]-2(3H)-furanone, known as HPMF. These may be present in free or conjugated form.
The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulfuric acid semi-ester sodium salt (abbreviated hereafter as “sulfate salt”). The conjugated estrogen content (calculated as estrogen sulfate salt) may be between 0.1 and 1% by weight, relative to the dry matter. In addition, further lipophilic compounds may be present in the solids content of the PMU, the quantities of which compounds can fluctuate within wide ranges and cannot be predicted. These lipophilic compounds originate predominantly from the plants ingested as food by the pregnant mares and primarily comprise various flavonoid, isoflavonoid and norisoprenoid derivatives and comparable compounds, such as for example formononetin, genistein, daidzein, biochanin A, equol and coumestrol. These lipophilic compounds originally of plant origin may be present in the urine in conjugated or in free (non-conjugated) form. The lipophilic constituents furthermore occurring in the solids content of the PMU also include non-conjugated steroid derivatives; notably including in particular the androstane and pregnane steroids and also non-conjugated estrogen derivatives.
Extracts containing natural mixtures of conjugated estrogens are usually obtained either by a solid-phase extraction method or by a method based on various liquid-liquid extraction steps with organic solvents which are not miscible, or only slightly miscible, with water. Generally speaking, in order to be able to be used as active substance constituent for pharmaceuticals, the natural mixture of conjugated estrogens which is obtained must meet certain pharmaceutical specifications, for example, the specification laid down in the USP (United States Pharmacopeia) or European Pharmacopoeia. For example, certain limit values must be observed with regard to the content of conjugated estrogens relative to the dry matter.
U.S. Pat. No. 2,551,205 and U.S. Pat. No. 2,429,398 describe a process for the preparation of a water-soluble estrogen preparation from PMU, in which initially an aqueous concentrate is obtained by adsorption on activated carbon or other suitable adsorber materials, elution with a water-miscible organic solvent, such as pyridine, and subsequent removal of the solvent, thereby yielding a concentrate which contains the major part of the water-soluble estrogen constituents of the original PMU. Whereas in U.S. Pat. No. 2,429,398 the concentrate is further purified by extraction with benzene and/or ether, U.S. Pat. No. 2,551,205 discloses acidulating the concentrate to a pH value of between 2 and 6, preferably between 4 and 5, and then rapidly extracting it with an organic solvent which is only slightly miscible with water selected from aliphatic, aromatic or alicyclic hydrocarbons (e.g. hexane, benzene, toluene, cyclohexane) or the chlorinated hydrocarbons (e.g. chloroform, ethylene dichloride, trichloroethylene, carbon tetrachloride, chlorobenzene), in order to separate undesirable substances such as fats, oils, free phenolic constituents and the non-conjugated steroids by transfer into the organic phase. Finally, the aqueous phase is stabilized by neutralization. U.S. Pat. No. 2,551,205 recommends further purifying the resulting extract by subsequent extraction steps and precipitation operations. Overall, after performing the method described in U.S. Pat. No. 2,551,205, a yield of only about 80% of the estrogen constituents of the concentrate used is obtained.
U.S. Pat. No. 2,565,115 describes the extraction of the conjugated estrogens from PMU with acetone. No statement is made about the purity of the resulting estrogen fraction.
U.S. Pat. No. 2,696,265 describes a method in which initially the estrogens are extracted with an aliphatic alcohol or ketone, such as hexanol, cyclohexanol or cyclohexanone. The estrogens pass into the organic phase and are then further purified; inter alia, an aqueous phase containing the estrogens is adjusted to a pH value of 4 with hydrochloric acid and extracted with ethylene dichloride.
U.S. Pat. No. 2,834,712 discloses a method for the preparation of estrogen mixtures of significant purity and low toxicity which is based on a large number of individual extraction steps with different solvents and the setting of different pH values. In that method, large volumes of solvents such as hexane and benzene are used. Thus, for example, in one step an already purified concentrate is dissolved in water, adjusted with hydrochloric acid to a pH value of approximately 5.0 and extracted with benzene and then with ether, in order to separate the phenolic constituents.
International patent application WO 01/27134 describes a comparatively simple method of extracting conjugated estrogens from PMU: after the addition of a salt, such as sodium chloride, the PMU is extracted with at least the same volume percent of an organic solvent, such as ethyl acetate, whereupon the conjugated estrogens pass into the organic phase. The organic phase is separated and dried in order to obtain the extract. No statements are made in WO 01/27134 about the purity of the conjugated estrogen extract which is obtained.
With the liquid-liquid-extraction method described above and known from the prior art, however, a number of problems occur, such as vigorous foaming, sediment formation, emulsification and poor phase separation. Generally several extraction steps are required, which results in losses and only partial recovery of the estrogen content. Furthermore, these extraction methods require large volumes of solvents some of which are harmful to health. Furthermore, in the patent specifications listed above no statements are made either about the content of non-conjugated lipophilic constituents, such as for example non-conjugated flavonoid, isoflavonoid and norisoprenoid derivatives and comparable non-conjugated compounds, or also non-conjugated steroids, in particular androstane and pregnane steroids, in the products obtained, nor about separation of these constituents. These methods known from the prior art either provide no satisfactory results with regard to the yield or with regard to the purity of the extract obtained, measured by the total hormone content obtained relative to the dry matter, or they are based on a large number of different method steps and the use of large volumes of organic solvents some of which are undesirable from an environmental or toxicological point of view.
Furthermore various solid-phase-extraction methods are known from the prior art for obtaining a natural mixture of conjugated equine estrogens largely depleted in phenolic urine contents. Thus international patent application WO 98/08526 describes a method with which a largely cresol- and HPMF-free mixture, which is depleted in phenolic urine content and contains practically the entire natural estrogen content of the PMU, can be obtained in a solid-phase extraction on a semipolar, in particular non-ionic semipolar, polymeric adsorption resin. International patent application WO 98/08526 describes a similar method in which silica gel is used as the adsorber material in the solid-phase extraction. Also Chinese patent application CN 1308083 describes a comparable method in which polar adsorption resins containing cyano groups are used. The extracts obtained are suitable as starting material for the preparation of pharmaceuticals which contain the natural mixture of conjugated estrogens from PMU as active substance constituent.
The established pharmaceutical specifications, for example, the limits to be observed regarding the content of conjugated estrogens relative to dry matter, are normally met by the mixtures of conjugated estrogens obtained from PMU in accordance with the method of WO 98/08526 or the method of WO 98/08525. It has, however, turned out that in addition to the desired content of conjugated estrogens also non-conjugated lipophilic compounds may be contained in the dry matter obtained. The non-conjugated lipophilic compounds include, for example, various non-conjugated flavonoid, isoflavonoid and norisoprenoid derivatives and comparable non-conjugated compounds, such as, for example, formononetin, genistein, daidzein, biochanin A, equol and coumestrol, but also non-conjugated steroids, in particular androstane and pregnane steroids, and non-conjugated estrogens. This list should not be regarded as exhaustive, however, since other non-conjugated compounds also may be present. The presence of the non-conjugated lipophilic compounds in the mixture of conjugated estrogens obtained from the PMU cannot be standardized, but both the content and the composition of the free and conjugated lipophilic compounds varies, for example, depending on the food ingested by the pregnant mares.
Although the composition of the natural mixture of conjugated equine estrogens does not change due to the additional presence of the non-conjugated lipophilic compounds, the content of the conjugated equine estrogens relative to the dry matter can be reduced. A higher concentration of the active substances, i.e. the conjugated equine estrogens, in the extract obtained could be achieved by deliberate separation of the non-conjugated lipophilic constituents. Also for reasons of medicament safety it may be useful to remove the non-conjugated lipophilic compounds in order to ensure a uniform composition of individual extract batches, since in this way the non-conjugated lipophilic constituents, the content and composition of which in the PMU can vary according to the seasonally changing type of food ingested by the pregnant mares, can be eliminated, and thus the resulting extracts would all have a comparable content of conjugated equine estrogens relative to the dry matter. Furthermore, separation of the non-conjugated lipophilic compounds may be advantageous in order to obtain a uniform physiological spectrum of action. For example, it may be useful to separate possibly present, non-conjugated lipophilic compounds, which may possibly themselves have an undesirable physiological effect, from the natural mixture of conjugated equine estrogens.